The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we are using a unique approach in which the centrosome (labeled with GFP) is ablated by laser micro beam. This approach allows us to create cells that lack the centrosome, and compare their behavior with genetically identical cells that possess this organelle. Further, we combine laser microsurgery with inactivation of individual centrosomal proteins by siRNA. This synergistic methodology provides new information not obtainable by other means. Our current research focuses on the two phenomena that we described during past years of the grant. First, we discovered that the presence of the centrosome is required in normal but not in cancerous cells to progress through the cell cycle during interphase. We also demonstrated that cancerous cells born without centrosomes re-form this organelle de novo. We now investigate particular molecular mechanisms that coordinate cell cycle progression and activation of the centrosome de novo formation pathway. For that we ablate centrosomes in human lines at different stages of transformation (e.g., deficient in p53, pRb, or p21) to determine which of these pathways is needed to block cell cycle progression in the absence of centrosomes (Aim 1). We also ablate centrosomes in cells depleted (by siRNA) of different centrosomal proteins to determine how individual proteins affect centrosome de novo formation (Aim 2). Our second line of research is to clarify the role of the centrosome in mitosis. We have demonstrated that centrosomes are not essential for the formation of mitotic spindles, however, mitosis in acentrosomal cells is error-prone. We now investigate the relative contributions of the centrosomal and centrosome-independent pathways for mitotic spindle formation. Aims 3 and 4 test two specific hypothesis: 1. That the centrosomes are required for efficient correction of synthetic chromosome mal-orientation;and 2. That the initiation of kinetochore fiber formation occurs via capture of short microtubules nucleated in the vicinity of kinetochores by centrosome-independent mechanisms. Since centrosome abnormalities are a hallmark of malignant tumors, the knowledge obtained in these studies will be useful for designing new strategies for the treatment of cancers.